Medical device for treatment of a gap or defect in the central nerve system

ABSTRACT

A medical device ( 1 ) of a biocompatible material for use in the treatment of a gap or defect in the central nervous system, which device has a proximal end ( 5 ) and a distal end ( 6 ) comprising openings ( 7 ). The device is adapted to enable connection of nerve fibers of gray and white matter between the proximal end ( 5 ) and distal end ( 6 ) thereof in predetermined openings ( 7 ). The device is of a substantially cylindrical form, or a substantially flat or plate like form and is made of plastic. The openings ( 7 ) in at least one end ( 5, 6 ) bear distinctively different indicia thereby to indicate whether nerve fibers of gray matter or nerve fibers of white matter are to be inserted therein.

FIELD OF THE INVENTION

The present invention relates to a medical device of a biocompatiblematerial for use in the treatment of a gap or defect in the centralnervous system.

BACKGROUND OF THE INVENTION

Treatment that promotes functional regeneration across a complete spinalcord transection in man does not exist. In animal experiments (1),recovery after incomplete spinal cord lesions has been achieved inadults treated with myelin-associated protein antibodies while recoveryafter complete lesions has been demonstrated in neonates (2).

Various attempts have been made over the years to find a replacement fordirect nerve stump to nerve stump suturing. Much research has focused onthe use of channels or tubular prostheses which permit the cut ends ofthe nerve to be gently drawn into proximity and secured in place.

All prostheses produced so far are intended for peripheral nerves suchas U.S. Pat. No. 3,833,002, U.S. Pat. No. 4,759,764, U.S. Pat. No.4,870,966 and SE 457 598.

SUMMARY OF THE INVENTION

The present inventors have now found that mobility can be restored inrats having complete spinal cord gaps. The gaps have been bridged withmultiple intercostal nerve grafts redirecting specific pathways fromwhite to grey matter, and the grafted area stabilised by acidicfibroblast growth factor-containing fibrin glue and compressive wiringof posterior spinal processes. Nerve bridges were created between theperipheral nerves and the spinal cord.

It has also turned out that motility can also be resored under certaincircumstances when grafting white to white and grey to grey mass ifspecial antibodies are given to the person being treated or incorporatedinto inserted material connecting the gap ends of the spinal cord.

The invention relates to a medical device of a biocompatible materialfor use in the treatment of a gap or defect in the central nerve systemcomprising one or more means containing connections such as holes orchannels arranged for receiving nerve growth promoting materials orsubstances, which device has a proximal and a distal end area comprisingopenings from the connections, characterized in that the device ismarked and arranged for coupling nerves from the grey and white matterof the central nervous system at the proximal end to certain markedareas in the proximal end area and nerves in the grey and white matterat the distal end to certain marked areas in the distal end area.

The device may be marked and arranged for coupling nerves from the greymatter of the central nervous system at the proximal end to nerves inthe grey matter at the distal end and nerves in the white matter of thecentral nervous system at the proximal end to nerves in the white matterat the distal end. This is especially the case when antibodies areadministrated in order to promote the nerves to grow together.

In a preferred device according to the invention the device or the meansis marked and arranged for redirecting nerves from the grey matter ofthe central nervous system at the proximal end to nerves in the whitematter at the distal end and nerves in the white matter of the centralnervous system at the proximal end to nerves in the grey matter at thedistal end.

The device can be used in the treatment of complete or partial gaps ordefect in the central nervous system. It may consist of one or moremeans comprising holes or channels arranged for receiving nerve growthpromoting materials or substances, which device has a proximal and adistal end area comprising openings from the holes or channels. Theopenings and/or the end areas may be marked for easily coupling nervesin the grey matter of the central nervous system at the proximal end ofthe gap to nerves in the white matter of the central nervous system atthe distal end and nerves from the white matter in the distal end tonerves in the grey matter of the proximal end.

The expressions proximal and distal refer to direction to and from thehead respectively of the individual receiving the transplant.

Preferably the device redirects descending motor pathways from proximalwhite to distal grey matter, and ascending pathways from distal white toproximal grey matter.

The device may consist of one means that has a substantially cylindricalform comprising channels bridging openings in a first part of the areaof the proximal end and openings in a second part of the area of thedistal end and channels bridging openings in the second part of the areaof the proximal end and openings in the first part of the area of thedistal end.

The end area can have any suitable form and be planar or non planar suchas curved or spherical.

In another embodiment of the invention the device may comprise one ormore means having a substantially flat or plate like form. Tubes ofbiocompatible or biodegradable material containing growth promotingmaterial and/or Schwans cells and/or one or more peripheral nerves arethreaded through the holes of the plate or plates. When using a nervefibre or bundles of nerve fibres the tubes may not be needed.

It is preferred to use as many fine nerves as possible. In theexperiment with rats described below 18 peripheral nerves were used tobridge one gap of the spinal cord. It has turned out that it is possibleto use even more nerves. Therefore it is postulated that up to 200nerves may be used on humans, such as 25-150, preferably 50-100 nerves.

It is also possible to use a device having one or more means withsubstantially cylindrical form and one or more means having asubstantially flat or plate like form. One means with cylindrical formcan be surrounded by one or more means with plate like form.

The means may also be one or more tubes of a biocompatible material tobe filled with the growth promoting substance and put in place in thespinal cord. The invention also relates to the use of such tubes. Thedevice and means may also have other forms and can e.g. be moulded tosuit the subject to be treated

When plates only are used they may be threaded with bundles of nerves orwith tubes of the biocompatible material filled with thegrowth-promoting substance. Thus the device may also comprise one ormore plates and tubing to be filled when used.

In order to facilitate the redirection of the grey and white matter, theareas of the proximal and distal ends respectively of the means aredivided in a first and a second part. The first and the second areas mayhave any form. It is possible to simply divide the end area in twoparts, that may be of the same or of different size. Preferably thefirst area is a central area and the second area is a peripheral areasituated substantially around the central area. It is however alsopossible that the areas have one or more parts thereof intermingling orsticking into each other with or without connection(s) to the main partarea.

Preferably the device consists of one cylindrical means having a firstperipheral part and a second central part of the proximal and distal endarea connected to the white and grey region respectively of both ends ofthe gap. Channels going from the peripheral part of one end are beingredirected to end up in the central part of the other end of thecylinder and vice versa.

In order to enhance long distance regeneration it is preferable to letone or more channels or bundles of nerve fibres pass the gap and insertthe end of the nerves or channels further down the spinal cord.Therefore the device or the means may have some of the channels endingat the side area and not at the distal end area. For example a nerve isintroduced in the white matter of the proximal end of the gap and ledthrough a channel that opens up in the side area of the device. It isthen introduced through the white matter further down beyond the gapinto the grey matter.

The means of the device may be marked. The first part of the proximalend area may be marked in the same way as the first part of the distalend area as is shown in FIGS. 1a and 1 b.

When the device comprises one cylindrical means the first (peripheral)part of both end areas can be marked in a different way from the second(central) part, e.g. white and grey to correspond to the colours of thespinal cord. Marking may not be needed when the device has a transverseend area and first and second parts of the same size as the white andgrey zones of the transverse area of the spinal cord.

In another embodiment the first part of the proximal end area of a meansis marked in the same way as the second part of the distal end area andthe second part of the proximal end area is marked in the same way asthe first part of the distal end area as is means 21 in FIG. 2. Thismarking may be practical when only one plate is used. It is, however,also possible to use one or more plates without marking them.

The grey matter lies in the centre of the spinal cord in the form of athick H or a butterfly surrounded by the white matter. There will beless crossing over of bundles of nerves or of channels if the first andsecond areas are more or less in conformity with the pattern of whiteand grey matter respectively in the CNS. Moreover there will be stillless crossing over if the switches between the different areas takeplace in the same halves or region of the cross section of the means.Thus, the switches are especially made in the same left (FIG. 4E) orright side region of the spinal cord.

The device may be marked e.g. by a vertically directed line (e.g. whenthe device has a plate like form) or an area (e.g. when the device has acylindrical form) in order to make the switches from white to greymatter in substantially the same left or right part of the spinal cordas was done in the rat experiment (FIG. 4E).

When two means in the form of plates are used they may be arranged asfollows. A first plate is intended to be placed near the proximal end ofthe gap and a second plate is to be placed near the distal end of thegap. The proximal and distal end area of the first and second plate hasdifferently marked first and second parts e.g. a white first area and agrey second part in both plates (see FIG. 3). The openings in the firstpart of the distal end area of the first plate and the openings of thesecond part of the proximal end area of the second plate are marked withfigures e.g. 1 to n. The openings in the second part of the distal endarea of the first plate and the openings of the first part of theproximal end area of the second plate are marked with different figurese.g. n+1 to n+m.

The marking may be in the form of different biocompatible colouringsubstances or different patterns.

According to the invention it is also possible to combine redirectingwhite to grey mass with direct coupling white to white and grey to greymass using special antibodies. Such a device may contain isolated areasin the first part marked in the same way as the second part and viceversa, in order not to redirect only some nerves that are coupled to theisolated areas using the antibodies.

The growth promoting material may be any pharmaceutically acceptablematerial or substance making the grey or white matter preferably thewhite matter grow. It may be a nerve growth enhancer such as for examplea growth factor or active analogue, fragment or derivative thereof Oneor more fibres, such as a bundle of numerous fibres or axons of aperipheral nerve and/or Schwans cells may be used. The nerves may beboth monofascicular and polyfascicular. Also mixtures of the abovementioned materials and substances can be used possibly together with aglue, that does not negatively affect the growth of nerves. The materialmay come from the individual to be treated or from other individualsfrom the same or some other species.

Preferably nerves coming from the ribs such as intercostal nerves aretaken out from the individual to be treated and cut into pieces. Thesenerve pieces or parts thereof are inserted in the holes or channels inthe device. It is preferred to have as many nerves as possible as moreand better functions are likely to be restored the more bridges that arecreated. One can use 1 to 100, preferably 9 to 75 especially 15 to 40bundles of nerve fibres.

The holes and the channels may have any transverse cross sectional formsuch as round, oval or square. They are preferably generally tubularwith round or oval lumen.

The diameter may be from 1 μm to 5 mm. Preferably visible bundles ofperipheral nerve fibres are inserted in the channels that have adiameter of about 0.2 mm-3 mm, especially 0.5 mm-2 mm.

The device can be delivered without any growth promoting material and beused together with e.g. peripheral nerves or Schwans cells from theindividual to be transplanted. It is however also possible to deliverthe device with a growth promoting material put in place possiblytogether with pharmaceuticals or substances preventing microbial andimmunological influence on the material during transport and storage orin the patient body. The device may also contain growth factors such asbioactive neotrophic factors e.g. aFGF incorporated into the material ofthe device or in the holes and/or channels. The growth factor may bepresent in a gradient concentration that may increase in the proximal ordistal direction.

Different gradients can be used for the space holding nerves coming fromthe white and grey matter respectively.

The device and the means may have any form. The transverse sectionalarea may for example be round, oval, square or rectangular. Preferablyit has about the same form and area as the transverse section of the gapin the spinal cord of the individual receiving the implant. The size ofthe individual affects the size of the spinal cord. Further the spinalcord is thicker in the regions where the nerves to the arms and legscome out. It is also possible to treat a complete or partial gap. Takingthis into account, and with regard to the fact that the means may beplate like or have the form of a cylinder, the device may be produced indifferent sizes. The length in the direction of the spinal cord may beabout 0.2 cm to 5 cm, preferably 0.5 cm to 4 cm. The length of thedevice depends on the length of each means that it is composed of. Thetransversal area of the device and means may vary from about 0.3 cm² toabout 4 cm².

The invention also relates to a method for restoring a deficiency in thespinal cord of humans comprising filling a device of a biocompatiblematerial with nerves and/or Schwans cells, possibly also growthpromoting material and possibly also a biocompatible glue and connectingwhite to grey mass of the spinal cord. When special antibodies are usedit is also possible to couple the nerves from white to white mass andfrom grey to grey mass.

It is also possible to combine the two different ways of coupling themass of the central nerve system and couple some of the nerves to thesame sort of mass (i.e. white to white and grey to grey) and some othernerves from white to grey.

The device may be composed of any biocompatible material such as, forexample, polyethylene vinyl-acetate (EVA); or of biocompatiblehydrogels, such as polyvinyl pyrolidone, polyethylene oxide (PEO),polyurethanes, acrylates, or mixtures thereof. Preferable acrylatesinclude methacrylates or hydroethylmethacrylates.

Alternatively it may be composed of a bioresorbable, or bioabsorbablebiocompatible polymer, such as a polyanhydride, polyester, or mixturesthereof; Poly-alpha-hydroxy acids (PGA); polylactic acid, copolymers oflactic and glycolic acids, and said polymers copolymerized with otherpolyesters such as epsilon—caprolactone; copolymers having a glycolicacid ester and trimethylene carbonate linkages, e.g. the copolymer inthe MAXON (American Cyanamid Company, Wayne N.J. 07470, USA) suture;polydioxanone; polyesters formed from diols and succinic and/or oxalicacid, isomorphic copolyoxalates and poly(alkylene) oxalates; polymersmade from unsymmetrically-substituted 1.4-dioxane-2.5-diones.

One can also use silicone, connective tissue fibres such as collagen,polyglycolic acid, composite made of collagen and glycosaminoglucan (seeU.S. Pat. No. 4,280,954).

The material of the device is preferably permeable to body liquids andsubstances in order to facilitate e.g. blood vessels growing into thedevice. The material shall have qualities in order to hold and keep thenerve fibres in place. Connective tissue fibres such as collagen aresuitable.

The device may be produced by using flexible tubes around which thebiocompatible material is moulded. The flexible tubes are bent to changethe direction of the channels from the first area of the proximal end tothe second area of the distal end and vice versa to create channelschanging direction from white to grey matter. The tubes are then drawnout from the device.

Devices, such as plates intended for changing the direction from whiteto grey outside the means or between the means, can be produced asdescribed in WO 90/05552.

The device is preferably delivered with a thread inserted in thechannels. When used, bundles of nerves are tied to the thread and drawnthrough the channels.

When used the device comprising nerve bundles or channels ofbiocompatible material containing Schwans cells and/or other nervegrowth promoting material are treated with a biocompatible gluecontaining one or more growth factors.

The glue to be used according to the invention is preferably a fibringlue containing 50-200 mg/ml, preferably 100 mg/ml of fibrinogen and 0.2μg-20 μg per ml especially 1 μg-5 μg per ml, especially 2.1 μg/ml ofaFGF (acidic fibroblast growth factor).

The fibrin glue may be a fibrinogen based compound with double sealantcomponents (Beringplast R P, Behring, Behringwerke A G, Margburg,Germany), containing a vial A with fibrinogen concentrate consisting of115-232 mg dry substance, containing a human plasma protein fractionwith 65-115 mg fibrinogen and a human plasma protein fraction with afactor XIII activity of 40-80 U, and a vial B with aprotinin solutionconsisting of 1 ml solution containing 1000 KIU bovine lung aprotininand a vial C of thrombin consisting of 4.9-11.1 mg dry substancecontaining a human plasma protein fraction with a thrombin activity of400-600 IU and a vial D with a calcium chloride solution consisting of2.5 ml solution containing 14.7 mg calcium chloride 2H₂O (40 mmol).

The advice of the supplier is followed and the aFGF is added to the vialA when used.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the figures ofwhich:

FIG. 1a is a perspective view of one embodiment of the invention and theends of the central nervous system at a gap, where the pathways in thecentral nerve system are redirected,

FIG. 1b is a perspective view of another embodiment of the inventionwhere the pathways are recoupled from white to white and from grey togrey mass,

FIG. 2 is a view from above of a device according to the inventionconsisting of two plates,

FIG. 3 is a view from above as in FIG. 2 showing possible marking of theplates with figures in order to facilitate the threading of nervesthrough the plates.

FIGS. 4A-F show the results from grafting of rat spinal cord, 4A-C showmicrophotographs illustrating stainings of sections through spinal cordsegments, 4D hindlimb function, 4E schematic illustration ofwhite-to-grey matter nerve bridges and 4F summary of data from animalsand

FIGS. 5A and 5B show morphology and sequential videoframes fromexperimental animals.

In the device 1 a of FIG. 1a the means 2 has the form of a cylinder witha proximal end area 5 and a distal end area 6 having first part 8 andsecond 9. The first part of the proximal and distal ends 5 and 6 arewhite and shaded respectively. The device contains channels 4 of whichthree are shown in the drawing. One channel 4 leads from the first part8 of the proximal end area 5 to the second part 9 of the distal area 6.Another channel leads from the second part 9 of the proximal end area 5to the first part 8 of the distal end area 6. One of the channels 4 endsin an opening 7 on the side area 14 of the device in order to be shuntedpassed the distal end of the CNS gap and introduced into the CNS furtherdown at a suitable angle through the white into the grey matter. Thechannels have been threaded with bundles of nerves (not shown).

The end areas have parts marked in different ways making it easy to seee.g. that channels coming out from the white first part 8 of theproximal end area 5 are to be inserted into the white matter 11 of theend area of the proximal gap 10. Channels coming from the grey secondarea 9 in the distal end 6 of the device are inserted in the grey matter13 of the end area of the distal gap 12. Correspondingly other channelscoming out from the white first part 8 and grey second part 9 of the endareas 5 and 6 of the device are inserted into the mass of the end areasof the CNS gap having the same colour. With the device of FIG. 1descending motor pathways from proximal white were coupled to distalgrey matter and ascending pathways from distal white to proximal greymatter according to the arrows.

The device of FIG. 1b is intended to be used together with a materialsuch as certain antibodies, that can induce growth in the nervematerial. Therefore the pathways need not to be redirected. The devicecomprises one means 20 in which two channels 4 are shown to lead fromthe first part 8 of the proximal end area 5 to the first part 8 of thedistal end area 6. Another channel 4 is shown to lead from the secondpart 9 of the proximal end area 5 to the second part 9 of the distal endarea 6. Thus, no redirection is made. The device of FIG. 1b may alsohave channels ending in openings on the side area 14 to be shunted pastthe gap and introduced into the CNS further down (not shown).

FIG. 2 shows the proximal and distal end areas of a device 1 consistingof two means 21 and 22 in the form of two plates with holes 3 throughwhich bundles of nerves or tubes filled with growth promoting materialsuch as Schwans cells may be threaded according to the dashed lines. Theproximal and distal ends of the two plates are shown. The plates 21, 22are marked 1P and 2P respectively in order to be placed in the rightorder and with the proximal ends towards the head of the patient. Theproximal end 5 of the first plate 21 marked “IP” has a white first part8′ and a grey second part 9′ to show where to insert the tubes or nervebundles into the white and grey matter of the proximal CNS gap later on.The distal end 6 of the first plate is marked in order to direct thethreading of the nerves coming out of the first plate, the first part 8′being grey and the second part 9′ being white. The proximal part 5 ofthe second plate 22 is marked to receive the nerves from the right partof the first plate in order to change the direction from white to grey.Thus, the first part 8 of the proximal end 5 is white and the secondpart 9 is grey, the first part 8 and second part 9 respectively of thedistal end 6 of the second plate 22 being marked in the same way, whiteand grey. The plates may change places but in such a case the crossingover from white to grey area will not take place between the plates butafter the second plate. When used, glue is placed between the plates andat the site of insertion or in the spinal cord.

It is also possible to mark the first and second part of the proximalarea of the first plate white and grey as in FIG. 2 but instead ofmarking the areas of the distal end of the first plate and the proximalarea of the second plate openings 7 of the holes 3 in these areas may benumbered in order to direct the threading as shown in FIG. 3.

When making and numbering the holes it is possible to avoid or mininisecrossing of the tubes or nerve bundles and to keep the redirectionbetween white and grey matter in the same left or right part of thedevice and the spinal cord.

Tests showing Spinal Cord Repair in Adult Paraplegic Rats and PartialRestoration of Hindlimb Function (Science, Jul. 26, 1996, in press)

Complete spinal cord gaps in adult rats were bridged with multipleintercostal nerve grafts redirecting specific pathways from white togrey matter, and the grafted area stabilised by acidic fibroblast growthfactor-containing fibrin glue and compressive wiring of posterior spinalprocesses. Hindlimb function improved progressively during the first 6months as assessed by two generally accepted scoring systems. Thecorticospinal tract regenerated through the grafted area to the lumbarenlargement, as did several bulbospinal pathways. Together these datasuggest a possible repair strategy for spinal cord injury.

To avoid ambiguities, and to model the most severe clinical scenario westudied adult rats with complete surgical transection of the spinal cordincluding removal of 5 mm of the cord at T8. Histology of excised piecesof spinal cord demonstrated complete transection (FIG. 4A). We thenproceeded with a repair strategy (3).

We used peripheral nerve implants (4) to bridge the gap in the spinalcord (FIGS. 4B and C and FIG. 5B) and found that multiple fine nerveimplants (18 nerves to bridge one gap) gave better precision than theuse of fewer thicker nerves. To evade oligodendroglial proteins thatinhibit axon regeneration (5), we re-routed regenerating pathways fromnon-permissive white to permissive grey matter (6). The peripheral nervebridges thus redirected descending motor pathways from proximal white todistal grey matter, and ascending pathways from distal white to proximalgrey matter (FIG. 4E), taking into account the specific anatomy of ratdescending and ascending pathways (7). To stabilise the lesioned areaand the peripheral nerve bridges, the grafted area was filled with afibrin-based tissue glue that does not impair nerve fibre growth (8),and fixed the vertebral column in dorsiflexion by wiring (9).

Acidic FGF is a normal spinal cord constituent (10). Lacking a signalsequence, it is thought to be sequestered within cells, and releasedonly after damage. Consequently, FGF may be involved in repair (11). Italso decreases gliosis and enhances nerve fibre development in spinalcord grafts (12). Mixing aFGF into fibrin glue allows slow release (13).

Animals were followed over time for signs of functional recovery andrated by two independent blinded observers using the CBS (14) and theOFWS (15). Key responses were video-recorded. Hindlimb function inanimals subjected to the five-step procedure improved significantly,beginning three weeks after operation and continuing through the year ofobservation (FIG. 4D). Animals subjected to unilateral treatment alsoimproved, although to a lesser degree. Four different control groups (14transection only, 5 cord removal only, 3 white-to-white matter bridging,2 omissions of aFGF), did not improve (FIG. 4D). Improvement wasmanifest as appearance of a functional posture in hindlimbs (usuallyflexion at hips, then knees, then dorsiflexion at ankles). In controls,hindlimbs remained extended and externally rotated. Improvement wassymmetrical in 6 cases (28%), and asymmetrical in the rest. Locomotioninvolved trotting rather than ambling gait (FIGS. 5H-J). Hindlimbspartially supported body weight and movements were noted in the threemajor joints. Such movements were not seen in any of the control groups.We also found strongly statistically positive effects on contact placingscores of both groups with full treatment (FIG. 4D), suggesting thatfunctional recovery involves the corticospinal tract (2).

Anterograde wheat-germ agglutinin horseradish peroxidase (WGA-HRP)tracing from the sensorimotor cortices and retrograde WGA-HRP tracingfrom the lumbar enlargements were used to document fibre tractregeneration (FIG. 1F, 16). The spinal cord and brain were analysed andlabelled nerve cell bodies counted (17). Sections from the engraftedarea were analysed with cresyl violet or antibodies againstneurofilament and glial fibrillary acidic protein (GFAP) to illustratemorphology, nerve fibres in the bridges, and the degree of gliosis.Anterograde tracing is exemplified by one treated animal (FIGS. 4F, 5E,5F, 5G) with labelled fibres descending in the dorsal funiculus of theproximal stump, traversing the bridging grafts, and reaching grey matterof the proximal portion of the distal stump. Fibres descended at theinterface between the dorsal funiculus and dorsal grey matter and endedalong the dorsomedial aspect of the dorsal horn in regions adjacent tosubstantial gelatinosa in the lumbar enlargement. The labelled fibreswere thus found in areas corresponding to the pathway of the ratcorticospinal tract as shown in rodent cortical ablation studies (7). Inthe four other animals analysed by anterograde tracing (FIG. 4F),labelled fibres traversed the bridging grafts and were found in greymatter to T11, T11-12, T12 or L1-2. In four transected control animalsand in one animal subjected to white-to-white matter bridging plus aFGF,the corticospinal tracts did not reach the distal spinal cord stumps.Anterograde tracing of controls indicated no connections to the distalstumps (FIG. 4F).

Application of WGA-HRP to the lesioned lumbar spinal cord led tolabelling of motoneurons in layers III-V of hindlimb motor areasbilaterally in cortex cerebri in two of the three treated animals (FIG.5A). Labelled neurones were also observed in dorsal tegmentum, the lowerlimb areas of the red nuclei, reticular nuclei, and raphe nuclei in alltreated animals. A few raphe neurones (8 cells) were labelled in one ofthe three transected controls and one grafted animal without aFGF (56cells). Numerous raphe neurones were labelled in animals receiving thefive-step procedure. Thus a substantial number of regeneratingdescending axons, including the corticospinal tract and othersupraspinal neurones, appears essential for functional recovery. Animalswith improvement of hind limb function showed evidence of regenerationof both the motor initiation and the voluntary gait modificationcircuits to the spinal cord central pattern generator (18), suggestingthat these reconnections allowed animals to partially control theirhindlimbs. The degree of functional recovery appeared correlated to thedegree of regeneration of motor fibre systems (FIG. 4F).GFAP-immunohistochemistry demonstrated large cysts and wide GFAP-poorgaps between the cord stumps (4.7±0.86 mm, n=5) in C1 controls, whereasanimals receiving the full repair strategy had significantly shorterGFAP-poor gaps (0.7±0.19 mm, n=4; p<0.001, ANOVA). The bridge grafts inthe latter group displayed rich neurofilament immunoreactivity.Researchers have studied many different models of spinal cord injury inanimals. Incomplete spinal cord lesions such as hemisections,contusions, compressions, and different chemical or mechanical partiallesions have generated valuable information about reactive andcompensatory changes, but several of these models are less suitable forstudies of functional recovery caused by regeneration of cut axons. Wehave shown that our procedure can lead to a substantial degree ofstructural and functional recovery in the completely transected adultrat spinal cord, including regeneration of pyramidal tract axons,hindlimb movements and weight support. It remains to be seen to whichextent our technique is applicable to the chronic paraplegic state andto humans.

References

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3. Vertebrae T7-10 were exposed in adult 250-g female Sprague-Dawleyrats, under halothane (1.5%) anaesthesia (respiratory rate_(—)60/min,rectal temperature <3° C. below normal). Following T8 and T9 posteriorlaminectomies and bipolar cauterisation to control haemorrhage, a 5-mmT8 spinal cord segment was removed with microscissors (FIG. 4A).Eighteen intercostal nerves collected in Hanks' were used to reconnectand redirect pathways between the spinal cord stumps (FIG. 4E). Thefloor of the cavity between the spinal cord stumps was covered by a thingelfoarn layer. Minced pieces of peripheral nerve were used to fill theangle between the gelfoam bottom and the ventral-most (white matter)surface of the cut end of the distal spinal cord stump, producing aslanted floor allowing some physical support of those nerve bridges thatwent from deep (white matter) proximal sites to somewhat more dorsal(grey matter) distal sites. The cut ends of the intercostal nervebridges were held against, and attached to the semi-dry spinal cordstump surfaces. The two fibrin glue sealant elements (Beriplast P,generously provided by Behring, Germany) were prepared (see 8 & 13).aFGF (kindly provided by Drs Yihai Cao and Ralf Petterson, LudwigInstitute for Cancer Research, Stockholm) was mixed into the fibrinogenplus aprotinin solution. This solution was mixed with 10 μl thrombinplus calcium chloride to form an aFGF-containing (2.1 μg/ml glue) gluecast (final glue volume_(—)10 μl) in the engrafted area. The T7 and T10spinal processes were fixed in dorsiflexion with an S-shapedmonofilament surgical steel (DS-20, Ethicon) loop, fastened to thespinal column with non-absorbable circumspinal threads. In experimentalanimals, one group was subjected to unilateral redirection and aFGF inthe glue (URDaFGF), the other two groups were subjected to bilateralredirection and aFGF glue (BRDaFGF) using autografts (50%) mixed withallografts (I) or only autografts (II). Control animals were subjectedto transection at T8 (C1), or removal of 5 mm cord segment (C2), orgrafted with the same methods except HBSS replaced aFGF (BRD), orgrafted using only white-to-white matter connections (RBaFGF). Animalswere caged on thick soft bedding, with heating from below during thefirst postoperative days. Manual emptying of the bladder was performedtwice daily as long as needed. Antibiotics (Borgal, Hoechst, 15 mg/kg,subcutaneously) was injected once daily 7 days. Decubitus sores onhindlimbs were treated with iodine-soaked dressings. Animals were killedif severe sepsis (urinary tract infection), infected decubitus sores, orother wounds occurred. For the major experiments described here, themortality was less than 10%. Experiments were approved by the animalresearch ethical committee of Stockholm. Animals were sacrificed atdifferent time points for histological analyses but no earlier than onemonth after surgery to ensure complete degeneration of cut descendingfibres in the distal stump (9).

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8. H. Cheng, S. Almström, L. Olson, J. Neural Transplant. Plast. 5, 233(1995).

9. H. Cheng, L. Olson, Exp. Neurol. 136, 149-161 (1995).

10. R. Elde, et al., Neuron 7, 349 (1991); Y. Cao, Thesis, KarolinskaInstitute (1993); M. Koshinaga, H. Sanon, S. Whittemore, Exp. Neurol.120, 32 (1993).

11. P. McNeil, L. Muthukrishnan, E. Warder, P. D'Amore, J. Cell Biol.109, 811 (1989).

12. M. Giacobini, B. Hoffer, G. Zerbe, L. Olson, Exp. Brain. Res. 86, 73(1991).

13. H. Cheng et al., Exp. Brain. Res. 104, 199 (1995).

14. K. Gale, H. Kerasidis, J. Wrathall, Expt. Neurol. 88, 123 (1985).Percentage functional deficit as indicated by the Combined BehavioralScore (CBS) was used for behavioural evaluation. The CBS (except hotplate), ranging from 100 points (complete paralysis) to 0 (normal),consists of motor score (0-45), toe spread (0-5), righting reflex(0-15), extension withdrawal (0-5), placing reflex (0-5), climbing oninclined plane (0-15), and swim test (0-10).

15. M. Beattie, J. Bresnahan, In: Criteria for the assessment ofrecovery of function: behavioural methods, M. Brown, et al., Eds.(American Paralysis Association, Springfield, N.J., 1989), pp. 16-25; D.Behrmann, J. Bresnahan, M. Beattie, B. Shah, J. Neurotrama 9, 197-217(1992). Open field walking measured the animal's gross locomotor abilityduring a 5-min testing period. Two coded animals were observedsimultaneously to stimulate locomotor activity and scored according to amodified Tarlov scale, ranging from 0 (flaccid paralysis) to 5 (normalwalking). This scale was subdivided to more accurately reflect thepattern of recovery of locomotor function as follows: 0, no spontaneousmovement; 1, movement that is not reflexive, movement in the hip and/orknee, but not ankle; 2, movement of the limb in all three major joints;3, active support, uncoordinated gait or occasional short bouts ofco-ordinated gait; 4, co-ordination of forelimbs and hindlimbs in gait(trotting), walks on knuckles or medial surface of foot or few toedrags); 5, normal.

16. L. Heimer, in Neuroanatomical Tract-Tracing Methods, L. Heimer, M.Robards, L. Zaborszky, Eds. (Plenum Press, New York, 1981, 1989). Forretrograde HRP-tracing, six 0.1 μl 5% WGA-HRP (Sigma) injections weremade into the lumbar enlargement (L4-5, 25 mm distal to the distalgraft-host interface) and combined with an L6 transection to enhance HRPuptake. Positive and negative controls included injection of WGA-HRPwith/without colchicin blockage or an acute cut at T8 (FIG. 4F). Foranterograde HRP-tracing, a total of 2 μl 5% WGA-HRP was injected intothe sensorimotor cortces using five 0.2 μl injections per side. Positiveand negative controls are also given in FIG. 4F. Animals were perfused48 hours later (Ringer's solution, 2% paraformaldehyde, 10% sucrose).For immunohistochemistry of grafted areas, tissues were further treatedwith 4% paraformaldehyde for 2 hours and then with polyvinylpyrrolidone(Sigma) for 6 weeks to allow sectioning of soft and bony tissuetogether. Cryostat 14-μm sections for immunohistochemistry and 60-(brainand brainstem) or 90-μm (spinal cord) free-floating sections fortetramethylbenzidine histochemistry were collected.

17. M. Abercrombie, Anat. Rec. 94, 239 (1946). HRP-labelled neuroncounts required presence of the nucleus. The total number of neurones(N) equals n t/(t+d), where n is the number of nuclei of HRP-labelledneurones counted times the section period; t is the section thicknessand d the average diameter of counted nuclei.

18. S. Grillner, in Control of Posture and Locomotion, R. Stein, K.Person, R. Smith, J. Redford, Eds. (Plenum Press, New York, 1973), pp.515-535.

19. We dedicate this paper to Arne Nylander. Supported by APA, theSwedish MRC, USPHS grants, Taiwan Chin-Lin Fund and NSC. We thank thelate Jan Arvidsson for valuable tracing advice, and assistance fromSusanne Ahnström, Monica Casserlöv, Kjell Erlandsson, and Ida Engqvist.

Figure legends:

FIG. 4. (A) Microphotograph illustrating cresyl violet-stainedtransverse section through removed spinal cord segment. Bar: 250 μm. (B)Sagittal section of a repaired junction area stained with cresyl violet.Note nerve bridge grafts. Boxed area indicates the redirected fasciculusgracilis (upper) and dorsal corticospinal tract (lower) between theproximal (left) and distal stump (right). Bar: 1000 μm. (C) Adjacentsection immunostained with MAb to neurofilament protein 70,000. Numerousnerve fibres are present in the intercostal nerve bridges. Bar: 200 μm.(D) Hindlimb function in the 7 different groups (see 3). Hindlimbs wereassessed independently (except inclined plane test) to identifyasymmetric recovery. Animal scores were generated by averaging hindlimbscores. Open field walking (upper panel) measured the animal's grosslocomotor ability during a 5-min testing period. In the treated groups(BRDaFGF I & II), the scores improved significantly more than in thecontrols (C1+C2) from the third week on (Mann-Whitney). Unilaterallytreated animals (URDaFGF) improved to a lesser degree as seen 6 monthsafter surgery. Percentage functional deficit as indicated by the CBS isillustrated in the middle panel. Animals subjected to the five-stepprocedure improved significantly compared both to animals in which thespinal cord was only transected (C1) and animals with removal of a cordsegment (C2, ANOVA). Contact placing score (lower panel) indicatesfunctional recovery of the corticospinal tract and is significantlybetter in animals subjected to the five-step procedure than controlsuntil 6 months (Mann-Whitney). ****: P<0.0001; ***: p<0.001; **: p<0.01;*: p<0.05. (E) Schematic illustration of white-to-grey matter nervebridges. Pieces of intercostal nerves were used to reconnect andredirect pathways between the spinal cord stumps, TBVII, ThIX: seventhand ninth thoracic segment). For each redirected tract, one to threeintercostal nerves were used. (F) Summary of data from animals in whichboth open field walking scoring and HRP-tracing data were collected.Ranking tracing results from 0 (negative) to 10 (normal), the fillrepair animals differ significantly from controls (p<0.001,Mann-Whitney).

FIG. 5. Morphology and sequential videoframes from experimental animals.(A) Tetramethylbenzidine histochemistry of cortex cerebri (1 mmposterior to bregma) in a rat one year after the five-step procedure and48 hrs after retrograde WGA-HRP tracing from the lumbar enlargement.Labelled pyramidal cells are found in the hindlimb motor area. Bar: 100μm. (B) Cresyl violet cross-section of bridge area in a treated ratafter 4 months. Note 15 nerve bundles (*) and blood vessels (¤). Bar:600 μm. (C-G) Anterograde transport of WGA-HRP after injection intosensorimotor cortex in a normal rat (C, schematically depicted in D) anda treated rat 3 months after surgery (F and G, schematically depicted inE). Sagittal section of the bridge area (F) shows labelled fibrestraversing bridging grafts (*) and reaching the distal stump (right).Some tissue was lost during free-floating processing of section (¤).Transverse section of the lumbar enlargement (G) shows labelled fibresin the dorsal funiculus at the white/grey matter interface, and adjacentto substantial gelatinosa. Bar: 200 μm in C and G, 500 μm in F. (H-J)Sequential videoframes (interval between each frame: 0.12 sec) of 3 ratssubjected to complete five-step procedures. (H) Animal after 3 months.Note that hindlimbs can support body weight during forward stepping. Toedragging was absent in this animal. (I & J) Rats climbing a 45-degreeinclined plane one year (I) or 3 months (J) after surgery. Hindlimbspartially supported body weight and displayed movements in all threemajor joints. A trotting, rather than ambling or random movement of thefour limbs was noted (data not shown), although the hindlimb toesdragged during walking in I.

What is claimed is:
 1. A medical device of a biocompatible material foruse in the treatment of a gap or defect in the central nervous system,comprising: a proximal end; a distal end; marked openings between saidproximal end and said distal end adapted to enable connection of nervefibers of gray and white matter; and at least two means adapted toreceive nerve growth promoting substances, wherein an area of each ofsaid proximal and said distal ends is divided into a first part adaptedto enable connection of white matter of a spinal cord and a second partadapted to enable connection of gray matter of a spinal cord.
 2. Themedical device according to claim 1, wherein said second part is acentral area and said first part is a peripheral area situatedsubstantially around said central area.
 3. A medical device of abiocompatible material for use in the treatment of a gap or defect inthe central nervous system, comprising: a proximal end; a distal end;predetermined openings between said proximal end and said distal endadapted to enable connection to nerve fibers of gray and white matter,wherein channels bridge said openings in a first part of an area of saidproximal end and bridge said openings in a second part of an area ofsaid distal end, and wherein channels bridge said openings in a secondpart of said area of said proximal end and bridge said openings in afirst part of said area of said distal end.
 4. A medical device of abiocompatible material for use in the treatment of a gap or defect inthe central nervous system, comprising: a proximal end; a distal end;and predetermined openings between said proximal end and said distal endadapted to enable connection of nerve fibers of gray and white matter,wherein channels bridge said openings in a first part of an area of saidproximal end and bridge said openings in a first part of an area of saiddistal end, wherein channels bridge said openings in a second part ofsaid area of said proximal end and bridge said openings in a second partof said area of said distal end.
 5. A medical device of a biocompatiblematerial for use in the treatment of a gap or defect in the centralnervous system, comprising: a proximal end; a distal end; predeterminedopenings between said proximal end and said distal end adapted to enableconnection of nerve fibers of gray and white matter, wherein saidopenings are defined by a diameter and a length greater than saiddiameter and said openings create a side area in the length direction;and at least one channel having openings in said side area.
 6. A medicaldevice of a biocompatible material for use in the treatment of a gap ordefect in the central nervous system, comprising: a proximal end; and adistal end; and predetermined openings between said proximal end andsaid distal end adapted to enable connection of nerve fibers of gray andwhite matter, wherein said device is substantially flat or plate-like.7. A medical device of a biocompatible material for use in the treatmentof a gap or defect in the central nervous system, comprising: a proximalend; a distal end; predetermined openings between said proximal end andsaid distal end adapted to enable connection of nerve fibers of gray andwhite matter; at least two substantially flat or plate-like meansbetween said ends; and tubing arranged to receive growth promotingmaterial and threaded through holes in said means.
 8. A medical deviceof a biocompatible material for use in the treatment of gap or defect inthe central nervous system, comprising: a proximal end; a distal end;predetermined openings between said proximal end and said distal endadapted to enable connection of nerve fibers of gray and white matter;and a substantially cylindrical means and at least one substantiallyflat or plate-like means between said ends.
 9. A medical device of abiocompatible material for use in the treatment of a gap or defect inthe central nervous system, comprising: a proximal end; a distal end;and predetermined openings between said proximal end and said distal endadapted to enable connection of nerve fibers of gray and white matter,wherein said openings in at least one of said proximal or said distalends have distinctly different indicia to indicate whether nerve fibersof gray matter or nerve fibers of white matter are to be insertedtherein.